Hyaluronic acid (or hyaluronan, HA) is a high molecular weight copolymer of 1→3-β-linked N-acetyl-D-glucosamine-1→4-β-D-glucuronic acid from the glycosaminoglycans family of biopolymers with unusual rheological properties. Its physiological functions include the lubrication and protection of cells, maintenance of tissue structural integrity, and transport of molecules to and within cells. HA is found in the extracellular matrix (ECM) and plays an integral role in its organization and structure. Hyaluronan influences cellular proliferation and migration in developing, regenerating and remodeling tissues and in tissues undergoing malignant tumor-cell invasion (see, e.g., B. P. Toole S. D. Banerjee, Oligosaccharides reactive with hyaluronan-binding protein, monoclonal antibodies recognizing hyaluronan-binding protein, and use in cancer therapy, U.S. Pat. No. 5,902,795, 1999; S. Kumar, D. West, D. B. Rifkin, M. Klagsburn (eds.) Hyaluronic acid and its degradation products modulate angiogenesis in vivo and in vitro. In Current Communications in Molecular Biology; Angiogenesis: Mechanism and Pathobiology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 90–94, 1987.).
HA binds specifically to proteins in the ECM, within the cytosol and on cell-surface receptors. The prevalence of hyaluronan-binding proteins indicates the importance of HA recognition in tissue organization, proliferation and differentiation, growth factor activities, and the control of cellular adhesion and motility. HA's role extends to embryonic development, modulation of inflammation, stimulation of angiogenesis and wound healing, and morphogenesis.
A number of extracellular matrix and cellular proteins, the hyaladherins, have specific affinities to HA within the extracellular matrix. These include aggrecan, cartilage link-protein, hyaluronectin, neurocan and versican. Cellular hyaluronan receptors such as CD44 (CD=“cluster of differentiation”) and RHAMM (receptor for hyaluronate-mediated motility) are also known. Recent evidence implicates the CD44-HA interaction in cancer metastasis (for reviews, see Entwistle, J.; Hall, C. L.; Turley, E. A. J. Cell. Biochem., 61, 569–577, 1996; Bajorath, J. Proteins: Struct. Funct. Genet., 39, 103–111, 2000.). Melanoma cells expressing high CD44 levels show increased cell motility and metastatic potential compared to the same cell types that expressed low receptor levels (see e.g., Birch, M.; Mitchell, S.; Hart, I. Cancer Res., 51, 6660–6667, 1991.). The presence of specific HA cell receptors provides therefore potential uses in cancer diagnosis and therapy. Other biomedical uses include cataract surgery, osteoarthritis, and prevention of post-surgical adhesions. HA also displays useful wetting and moisture-preserving functions that are of interest in cosmetic and topical medical areas. HA sources include rooster combs, umbilical cords, shark skin, bull's eye and fermentation.
The integrin receptor family binds to ECM receptors (S. M. Abelda, Role of integrins and other cell adhesion molecules in tumor progression and metastasis, Lab Invest., 68, 4–17, 1993.). Integrins are heterodimeric glycoproteins with two subunits (α and β). A given β-subunit can pair with a number of α-subunits, resulting in various integrins with unique binding properties. Thus, α2β1 constitutes a collagen receptor that does not interact with laminin on platelets (C. J. Anderson, Bioconjugate Chem. 12, 1057–65, 2001.)
Normal human tissue cells express various integrins such as α1β1, α2β1, α3β1, and α6β1 that are required for adhesion to collagen and laminin (J. L. Lauer, C. M. Gendron, G. B. Fields, Effect of ligand conformation on melanoma cell alpha3beta1 integrin mediated signal translocation event Implication for a collagen structural modulation mechanism of tumor cell invasion, Biochemistry, 37, 5279–87, 1998.). Radiolabeled ECM fragments are useful imaging agents since their integrins are upregulated in certain tumors and can be targeted for diagnosis and therapy. Integrins promote adhesion, signal transduction and linkage between intracellular proteins and ligands. ECM fragments are used as imaging agents as their integrins are upregulated in certain tumor types and can be targeted for diagnostic or therapeutic use.
The ubiquitous nature of HA in biological systems, coupled with its antitumor and diverse range of other medical activities make diagnostic probe-carrying HA derivatives attractive for diagnostic and therapeutic uses. There is furthermore growing evidence that oligosaccharides derived from hyaluronan also bind to CD44. Thus, if an antagonist could be found for the CD44 receptor that would prevent HA binding, it would be possible consequently to limit metastasis. Such small molecules would have advantages over HA itself in that they would possibly be water soluble, membrane penetrating, and easy to administrate. Minimally, a 6-mer (hexasaccharide) is required for binding to CD44 and the 10-mer (decasaccharide) is required to displace HA from the HA-CD44 complex.
Other acidic polysaccharides, such as alginate and pectin are possibly also biologically active, as some evidence indicates in the literature (A. Kawada, N. Hiura, S. Tajima, H. Takahara, Alginate oligosaccharides stimulate VEGF-mediated growth and migration of human endothelial cells, Arch. Dermatol. Res., 291, 542–7, 1999; M. Sakurai, H. T. Matsumoto, H. Kiyohara, H. Yamada, B-cell proliferation activity of pectic polysaccharides from a medicinal herb, Immunology, 97, 540–7, 1999; H. Yamada, Contribution of pectins on health care, in J. Visser, A. G. J. Voragen eds., Pectins and Pectinases, Elsevier, Amsterdam, 173–190, 1996; H. Yamada, H. Kiyohara, Complement-activating polysaccharides from medicinal herbs, in H. Wagner ed., Immunomodulatory Agents from Plants, Birkhauser Verlag, Basel, 1999.). The preparation of alginate oligosaccharides (A, Martinsen, G. Skjak-Braek, O. Smidsrod, Carbohydr. Polym., 15, 171–173, 1991. Ikeda, H-F, A. A Takemura, H Ono, Carbohydr. Polym., 42, 421–425, 2000.) and pectic oligosaccharides (N. O. Maness, A. J. Mort, Anal. Biochem., 178, 248–254, 1989.) has been reported.
Hyaluronan has attracted considerable interest as biocompatible, resorbable material for tissue engineering and a wide range of other biomedical applications (for reviews, see D. Campoccia, P. Doherty, M. Radice, P. Brun, G. Abatangelo, D. F. Williams, Semisynthetic resorbable materials from hyaluronan esterification, Biomaterials, 19, 2101–2127, 1998; E. Milella, E. Brescia, C. Massaro, P. A. Ramires, M. R. Miglietta, V. Fiori, P. Aversa, Physico-chemical properties and degradability of non-woven hyaluronan benzylic esters as tissue engineering scaffolds, Biomaterials, 23, 1053–1063, 2002.) A considerable number of hyaluronan derivatives have been reported (see, e.g., K. P. Vercruysee, G. D. Prestwich, Hyaluronate derivatives in drug delivery, Crit. Rev. Therapeut. Carrier Syst., 15, 514–555, 1998; Y. Luo, G. D. Prestwich, Hyaluronic acid-N-hydroxysuccinimide: a useful intermediate for bioconjugation, Bioconjugate Chem. 12, 1085–88, 2001.). Collagen, the other major component of the extracellular matrix, constitutes over 30% of the human protein content and is associated with a number of diseases. Collagen has therefore been similarly widely employed as biocompatible matrix, as have hybrid materials derived from collagen and hyaluronan (S.-N. Park, J-C. Park, H. O. Kim, M. J. Song, H. Suh, Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking, Biomaterials, 23, 1205–1212, 2002). Collagen features an unusual amino acid composition: glycine constitutes over 30%, proline and hydroxyproline about 20%, whilst it lacks tryptophan and cysteine (i.e., no disulfide bonds).
Poly(glutamic acids), and in particular poly(γ-glutamic acid) (γ-PGA) are new biodegradable materials with many potential biomedical uses (I.-L. Shih, Y.-T. Van, The production of poly(γ-glutamic acid) from microorganisms and its various applications, Bioresource Techn., 79, 207–225, 2001). γ-PGA, elaborated by various Bacillus species (e.g., B. licheniformis), is an unusual polypeptide with its glutamic acid residues linked linearly through the γ-carboxyl function. γ-PGA assumes an a-helix conformation in solution, and, unlike the synthetic α-PGA analog, is a well-defined, high molecular weight homopolymer.
γ-PGA's polyanionic nature renders it highly water soluble and permits modulation of its solution conformation by co-solutes. PGAs ability to undergo conformational changes in response to different pH values offers the potential to affect targeted delivery. γ-PGA features a high molecular weight range and different solution conformations, is biocompatible, biodegradable (it biodegrades to glutamic acid monomers), non-toxic, and non-immunogenic nature. γ-PGA is also highly mucoadhesive, a key feature for localizing it site-specifically as a drug delivery vehicle in the small intestinal or colonic mucosa.
Radiolabeled peptide hormone analogues are of interest as diagnostic and therapeutic vehicles for treating cancer (Cutler C. S. Lewis J. S. Anderson C. J. Adv. Drug Deliv. Res., 37, 189–211, 1999. Anderson C. J. Welch M. J., Chem. Rev., 99, 2219–2234, 1999.; Anderson C. J. Dehdashti F Cutler P. D. Schwarz S W. Laforet R. Bass L. R. Lewis J. S. McCarthy D. W., J. Nucl. Med., 42, 213–2334, 2001.). These radiolabeled peptide receptor ligands can target upregulated cell surface receptors on tumors. For example, 111In-DTPA-octreotide is employed for imaging of neuroendocrine tumors that overexpress the somastatin receptor (E. P. Krenning, D. J. Kwekboom, W. H. Bakker, W. A. P. Breeman, P. P. M. Kooji, H. Y. Oei, M. van Hagen, P. T. E. Postema, M. de Jong, J. C. Reubi, T. J. Visser, A. E. M. Reji, L. L. J. Holland, J. W. Kuuper, S. W. J. Lamberts, Somatostatin receptor scintography with [111In-DTPA-D-Phe] and [111In-Tyr3]octreotide, Eur. J. Nucl. Med., 20,716–731, 1993.).
Primary human tumors from colon, ovary, skin and stomach and their metastatic sites show high levels of α3β1, and similarly cultured human cell lines (e.g., breast, ovarian carcinoma) express α3β1. Non-invasive means of monitoring α3β1 expression could be useful as a diagnostic tool for assessing metastasis prior to surgery. Since natural collagens are integrin ligands radiolabeled collagen fragments can serve as imaging agents.
There is a considerable demand for versatile non-invasive diagnostic probes, and fluorine's diagnostic value is of particular interest in non-invasive imaging applications. Apolar oxygen imparts paramagnetic relaxation effects on 19F nuclei associated with spin-lattice relaxation rates (R1) and chemical shifts. This effect is proportional to the partial pressure of O2 (pO2). 19F NMR can therefore probe the oxygen environment of specific fluorinated species in cells and other biological structures.
Nöth et al. (U. Nöth, P. Grohn, A. Jork, U. Zimmermann, A. Haase, J. Lutz, 19F-MRI in vivo determination of the partial oxygen pressure in perfluorocarbon-loaded alginate capsules implanted into the peritoneal cavity and different tissues, Magn. Reson. Med., 42(6), 1039–47, 1999) employed perfluorocarbon-loaded alginate capsules in MRI experiments to assess the viability and metabolic activity of the encapsulated materials. Quantitative 19F-MRI was performed on perfluorocarbon-loaded alginate capsules implanted into rats, in order to determine in vivo the pO2 inside the capsules at these implantation sites. Fraker et al. reported recently a related method with perfluorotributylamine (C. Fraker, L. Invaeradi, M. Mares-Guia, C. Ricordi, PCT WO 00/40252, 2000).
Although a large range of fluorinated products is available commercially, most PFCs suffer from a number of shortcomings. Many commercial PFCs currently employed for diagnostic purposes were originally selected for blood substitution. Their physicochemical properties [J. G. Reiss et al., Biomat. Artif. Cells Artif. Organs, 16, 421–430, 1988.] are therefore not targeted towards specific diagnostic or other biomedical uses, particularly for MRI. The molecular features of these PFCs are not optimized for high-sensitivity 19F-MRI studies. Their T1 relaxation times are relatively long, T2 relaxation times are short, and severe J-modulation effects and chemical shift artifacts can profoundly limit their MRI utility. Whilst their immiscibility in water offers benefits in some respects, it necessitates the use of emulsifiers. Thus, for PFC-in-water emulsions, such as F-44E, perfluorohexyl bromide (PFHB), perfluorooctyl bromide (PFOB, Perflubron™), perfluoromethyldecalin (PMD), perfluorooctyl ethane (PFOE), perfluorotripropylamine (FTPA), and the blood substitutes Fluosol™ and Oxygent™, lecithins or poloxamers are employed to disperse the PFCs and stabilize the emulsion. Fluosol™ was a 20% w/v mixture of 14% perfluorodecalin and 6% perfluorotripropylamine emulsified primarily with Pluronic F-68™. Oxygent™ is a 60% emulsion consisting mostly of PFOB and perfluoro-decylbromide, water, salts, and a lecithin. However, surfactants are problematic in that their use adds processing requirements and some of them can be unstable, chemically ill-defined or polydisperse, or cause potential undesirable side effects. Thus, Pluronic F-68™, the surfactant in Fluosol™, caused a transitory anaphylactic reaction in certain patients. Further, the stability of Pluronic F-68-based emulsions was limited; requiring frozen storage and mixing with two annex solutions prior to administration. The use of emulsions poses the additional disadvantage that the PFCs' fluorine content is effectively diluted (often by 50% or more), diminishing their spectral and imaging signal intensities and, hence diagnostic benefit. The impact of such dilutions is particularly evident in tumor oxygenation studies where only ˜10% of the injected PFC emulsion dose reaches the tumor, necessitating time consuming T1 measurements. This dilution effect is even more pronounced, when only a portion of the available PFCs' fluorine resonances is of diagnostic value. This is often the case, as severe chemical shift artifacts need to be circumvented by selectively exciting only a narrow chemical shift range containing one resonance (or a closely spaced group of resonances). Although F-44E, for instance, has a high fluorine content (74%) with largely acceptable spectral features, many MRI studies have selectively excited its trifluoromethyl resonance, representing only one third of the total F-content, which on emulsification (at 90%) is further diluted to ˜22%. Similarly, for MRI with perfluorononane the choice is between the selective acquisition of the single trifluoromethyl resonance (6 fluorines with a spectral width of 50 kHz at 7 Tesla) or multiple difluoromethylene resonances (14 fluorines with a 1300 kHz spectral dispersion) (see, e.g., S. L. Fossheim; KA ll'yasov, J. Hennig, A. Bjornerud, Acad. Radiol., 7(12), 1107–15, 2000.).
Ideally, PFC imaging agents should combine the following features: non-toxic, biocompatible, chemically pure and stable, low vapor pressure, high fluorine content, reasonable cost and commercial availability. Additionally, they should meet several 19F-NMR criteria, including a maximum number of chemically equivalent fluorines resonating at one or only few frequencies, preferably from trifluoromethyl functions. Some of the other spectral criteria have been discussed in detail elsewhere (C. H. Sotak, P. S. Hees, H. N. Huang, M. H. Hung, C. G. Krespan, S. Raynolds, Magn. Reson. Med., 29, 188–195, 1993.). For MRI, it would furthermore be desirable to have control over the amount of magnetically responsive material for specific uses, and to employ temperature-responsive and pH-dependent imaging agents for special uses. These could have applications in MRI-based temperature monitoring for use in general hyperthermia treatment (see, e.g., S. L. Fossheim; K. A. ll'yasov, J. Hennig, A. Bjornerud, Acad. Radiol., 7(12), 1107–15, 2000.) of tumors and for monitoring the efficacy of chemotherapy, respectively (see, e.g., N. Rhagunand, R. Martinez-Zagulan, S. H. Wright, R. J. Gilles, Biochem. Pharmacol., 57, 1047–1058, 1999; I. F Tannock, D. Rotin, Cancer Res., 49, 4373–4383, 1989.). Furthermore, water solubility would enhance the PFC functionality in many biomedical settings, as it would obviate the need for emulsifiers.
Although selected efforts have been directed at developing new fluorinated MRI probes, none are water soluble compounds [e.g., perfluoro-[15]-crown-5 ether)], and some are commercially unavailable [e.g., perfluoro-2,2,2′,2′-tetramethyl-4,4′-bis(1,3-dioxalane)-PTBD]. It appears no attempts have so far focused on screening available PFCs from the thousands of commercial fluorinated products in order to identify potentially more suitable MRI probes for biomedical uses. It seems furthermore that no studies have attempted to establish structure activity relations (SARs) of related PFCs for MRI purposes. Noteworthy is also the fact that all PFCs examined to date have molecular weights under 1,000, typically between 400–600 Da. This is partly a reflection of the specific requirements for blood substitution agents, but also due to the widely held belief that higher molecular weight or polymeric fluorinated agents would not be detectable by 19F-NMR due to anticipated excessive line broadening, and would therefore be unsuitable. Thus, with the exception of the polymer-encapsulated PFCs noted above, this important class of materials had so far been excluded from consideration.
Paramagnetic ions, such as gadolinium (Gd3+) decrease the T1 of water protons in their vicinity, thereby providing enhanced contrast. Gadolinium's long electron relaxation time and high magnetic moment make it a highly efficient T1 perturbant. Since uncomplexed gadolinium is very toxic, gadolinium chelate probes, such as gadolinium diethylenetriamine pentaacetic acid (GdDTPA MW 570 Da), albumin-GdDTPA (Gadomer-17, MW 35 or 65 kDa), have been employed extensively in MRI of tumors and other diseased organs and tissues. Several other developmental chelators have also been reported, including dual-labeled agents, oligonucleotide-derived, dextran-derived GdDTPA, and TAT and other peptide-derived chelators. However, presently approved MRI contrast agents are either not tissue specific, e.g., GdDTPA, or target only normal tissue, which limits their utility in diagnosis of metastases or neoplasia. MRI studies with GdDTPA, for instance, do not correlate with the angiogenic factor or the vascular endothelial growth factor (VEGF). Attempts have also been made to overcome the low relaxivities of small Gd-DTPA chelates by preparing polymer conjugates of Gd(DTPA)(2−) [see e.g., M. R. A. Duarte, M. G. Gil, M. H. Peters, J. A. Colet, J. M. Elst, L. Vander; R. N. Muller, C. F. G. C. Geraldes, Bioconjug. Chem., 21, 170–177, 2001.]. However, the relaxivity of these polymer conjugates was only slightly improved and they were also cleared very quickly from the blood of rats, indicating that they are of limited value as blood pool contrast agents for MRI.
Whilst much can be achieved with currently available imaging and contrast agents, there are still unmet needs for novel diagnostic agents, particularly for those exploiting biological specificity. Imaging agents suitable for targeting metastases or neoplasia would substantially enhance the MRI sensitivity and utility for tumor detection and prevention. Although selected efforts have been directed at developing such new probes, a broader investigation of these agents is urgently needed. Similarly, new imaging probes are needed as noninvasive means to detect and image cells, tissues and organs undergoing apoptosis. An even greater demand exists for biocompatible materials in tissue engineering and various other biomedical applications.